Stereo-Selective Oxidations of Terpinolene by Cytochrome P450 Monooxygenases in the Microsomal Fraction of Cupressus Lusitanica Cultured Cells

J Wood Sci (2014) 60:446–452 DOI 10.1007/s10086-014-1432-0

ORIGINAL ARTICLE

Stereo-selective oxidations of terpinolene by cytochrome P450 monooxygenases in the microsomal fraction of Cupressus lusitanica cultured cells

Takako Harada • Koki Fujita • Jun Shigeto • Yuji Tsutsumi

Received: 16 June 2014 / Accepted: 20 August 2014 / Published online: 13 September 2014 Ó The Japan Wood Research Society 2014

Abstract We showed previously that in Cupressus lusi- Keywords Cytochrome P450 Á Cupressus lusitanica Á tanica (Mexican cypress), the first two steps of terpinolene Microsomal fraction Á Stereo-selective oxidation Á Terpene oxidation, beginning at hydroxylation at the aryl position and then forming epoxide at the double bond, were driven by cytochrome P450s. The significance of enantio differ- Introduction ences, in general, has received attention because those enantiomers often have their own biological activities. We, Cytochrome P450s are ubiquitous in all kingdoms [1]. therefore, investigated the stereo-specificity of the substrate They have the functions of activating molecular oxygen and the enantio-selectivity of these reactions. The and introducing one oxygen atom into the substrate [2]. In hydroxylation of terpinolene by cytochrome P450 in the plants, this group of enzymes is involved in many sec- microsomal fraction from C. lusitanica cells gave a single ondary metabolisms, and all of the cytochrome-P450-pro- product with an S configuration of 5-isopropylidene-2- duced compounds can be expected to have significant methylcyclohex-2-enol. Next, epoxidizing enzyme accep- roles; for example, in helping plants survive or be com- ted only the S-configuration substrate and produced a sin- petitive in nature. Some of the plant secondary metabolites gle enantiomer product, (1R,2S,6S)-(?)-1,6-epoxy-4(8)-p- are also commercially attractive, and the details of cyto- menthen-2-ol. No isomer was detected at our gas chro- chrome P450 concerning biosynthesis have thus been matography/mass spectrometry sensitivity; therefore, the eagerly investigated [3]. calculated enantiomeric excess values were 100 %. These In particular, the significance of enantio differences has results indicate that the cytochrome P450s involved in received increasing attention over the past three decades terpinolene metabolism in C. lusitanica cells have very [4]. Each enantiomer has its own biological activities such strict stereo-selective ability. Our findings may be helpful as vasodilatory activity [5], antimicrobial activities [6], and in the stereo-selective synthesis of fine chemicals, although more as reviewed by Fassihi [7]. The topic of stereo- the physiological meanings of these chiral products are still chemistry in the fields of chemical ecology and medical not unclear. and biomedical studies has become important, and inves- tigations of substrate stereo-selectivity and enantio-selective products are now essential for plant metabolism research and for use in daily life. It is for these reasons that Parts of this report were presented at the 60th Annual Meeting of the plant cytochrome P450s have been examined for their Japan Wood Research Society, Miyazaki, March 2010, TEAC 2010, Kofu, October 2010 and TERPNET2011, Kalmar, Sweden, May specific substrate stereo-selectivity and enantio-selective 2011. products, although animal and fungal cytochrome P450s have relatively broader selectivities and reactivities [8]. T. Harada Á K. Fujita (&) Á J. Shigeto Á Y. Tsutsumi The lifespan of many woody plants should be tens or Department of Agro-Environmental Sciences, hundreds of years or more, and their stems are tall and Faculty of Agriculture, Graduate School of Kyushu University, Hakozaki 6-10-1, Higashi-Ku, Fukuoka 812-8581, Japan sound. These plants must keep fighting against many e-mail: [email protected] potential pests and pathogens, and they accumulate many 123 J Wood Sci (2014) 60:446–452 447 antibiotics in their tissue [9]. We have been investigating HO the biosynthesis of b-thujaplicin (hinokitiol) [10, 11]asa HO A monoterpene in Cupressaceae trees, which have a strong and a wide spectrum of antimicrobial activity [12]. How- R-IME S-IME ever, woody plants are not good research material for the study of metabolism and enzymes because they have very Ion intensity hard tissue and almost no living cells except at only the cambium and parenchyma. Therefore, our group has established an experimental 38 39 40 41 42 43 system using cultured cells obtained from Cupressus lusi- Retention time (min) tanica (Mexican cypress) [13]. The elicitor-treated C. lu- 109 sitanica cell cultures produce many types of olefine and oxygenated monoterpenes with a significant amount of 119 B 13 b-thujaplicin [14, 15]. In this cell line, a C-glucose 134 91 feeding experiment demonstrated that b-thujaplicin was 67 biosynthesized via an ordinary six-membered ring inter- 152 mediate, i.e., a menthane-type skeleton produced by terpene synthase and followed by ring expansion to a unique conjugated seven-membered ring, tropolone [16]. Assays 60 80 100 120 140 160 of terpinolene and terpinolene synthase suggested that 109 terpinolene is the first olefin monoterpene intermediate in C b-thujaplicin production [15]. 119 In fact, this speculation was confirmed by the results of a 134 feeding experiment using deuterized terpinolene added to 91 67

the cell culture [17]. It was also shown that the microsomal Ion intensity Ion intensity fraction from the cells oxidized terpinolene into the 152 hydroxylated compound, 5-isopropylidene-2-methylcyclohex-2-enol (IME), and then IME was further oxidized by 60 80 100 120 140 160 the microsomal fraction to the epoxidized compound, 1,6- 109 epoxy-4(8)-p-menthen-2-ol (EMO) [18]. Experiments on kinetics and with speciﬁc inhibitors conﬁrmed that these 119 D reactions were caused by cytochrome P450 monooxyge- 134 nases [18]. Typical cytochrome P450s can oxidize their 91 substrate with strict stereo-control as mentioned above, and 67 Ion intensity the EMO in the cultured cells was enantio-pure [14]. In the 152 present study, we investigated the substrate stereo-speci-

ﬁcity and enantio-selectivity of products on this metabolic 60 80 100 120 140 160 pathway. m/z

Fig. 1 Results of the chiral GC/MS of chemical synthesized IMEs and enzymatic produced IME. a Total ion chromatogram. Upper line Materials and methods chemically synthesized IME before chiral HPLC purification. Lower line enzyme product from terpinolene and the microsomal fraction Cell culture and microsome preparation prepared from C. lusitanica cultured cells. b The EI mass spectrum of the peak at retention time (Rt) = 40.7 of panel A upper. c The EI mass spectrum of the peak at Rt = 40.8 of panel A upper. d The EI Callus cultures of C. lusitanica were maintained in Gam- mass spectrum of the peak at Rt = 40.8 of panel A lower borg B5 medium [19] supplemented with 0.01 mM benzyl aminopurine, 10 mM naphthyl acetic acid, 20 g/L sucrose and 2.7 g/L gellan gum at pH 5.5 for more than 10 yrs. No 2 mL of partially purified yeast extract solution as an terpene including b-thujaplicin was produced under this elicitor [21]. After 24 h of 25 °C, 70 rpm shaking, and dark growth condition. For enzyme induction, approx. 5 g of C. incubation, cells were separated from the medium and lusitanica cells growing in solid B5 medium for 4 weeks stored at -80 °C. was transferred to 30 mL of liquid b-thujaplicin production For the preparation of the microsomal fraction, we medium [20]. To initiate terpene production, we added ground 8 g of the cells in a pre-chilled mortar and pestle 123 448 J Wood Sci (2014) 60:446–452 with liquid nitrogen, and suspended the powder in 200 mL Fig. 1. The absolute configurations of the secondary of pre-chilled buffer as described by Bouwmeester et al. hydroxyl group of IME were determined by a modified- [22]. The homogenate was transferred to a small bottle, Mosher method [24]. The results of this analysis are shown sonicated for 3 min in 10-s pulses at 35 % power (USP- in Fig. 2. 400A, Shimadzu, Kyoto, Japan) and stirred for 12 min. The homogenate was centrifuged at 20000 g for 20 min, fol- Preparation of authentic EMO lowed by filtration with a glass fiber filter, and the super- natant was then centrifuged at 150000 g for 90 min. Because EMO has already been discovered in extracts of C. Microsomal pellets were either used directly for the assays lusitanica cells and medium [14], we obtained authentic or stored at -80 °C. EMO by the extraction and purification of the cells and medium. We isolated EMO from the extracts by silica gel Preparation of authentic IMEs column chromatography. The purity of the obtained EMO was confirmed by chiral and achiral GC/MS analysis as We chemically synthesized authentic IME by Motherwell’s described above. method [23] as described by Harada et al. [18]. The purity and structure of the synthesized IME was confirmed by gas Microsomal reaction and enzyme product assay chromatography–mass spectrometry (GC/MS) (HP 5890 series II and HP 5972; Hewlett Packard, Palo Alto, CA) We conducted an assay of cytochrome P450s in micro- with the fused silica capillary column InertCap 5 (length, somal fraction as reported [18]. Microsomal pellets were 30 m; i.d., 0.25 mm; film thickness, 0.25 lm; GL Sci- resuspended in assay buffer [22]. One milliliter of micro- ences, Tokyo) was used. Helium was used as the carrier gas somal suspension was incubated in a 20-mL Teflon-lined with a column head pressure of 100 kPa. The oven tem- screw cap vial flushed with O2 gas, and the reaction was perature was kept at 70 °C for 3 min and increased with a started by the addition of 1 mM NADPH, an NADPH- gradient of 10 °C/min up to 250 °C. The temperature of the regenerating system (5 mM glucose-6-phosphate, 1 i.u. inlet port was 220 °C and that of the interface to MS was mL-1 glucose-6-phosphate dehydrogenase) and 550 nmol 250 °C. The purified products’ structures were determined of terpinolene or 315 nmol of IME (10 lL of acetone stock, by 1H- and 13C-nuclear magnetic resonance (NMR) spec- respectively). After incubation for 4 h at 30 °C, reactions troscopy (AL-400; JEOL, Akishima, Japan). Each of the were stopped by the addition of Et2O or ethyl acetate. As chiral compounds were enantio-purified by a chiral high- an internal standard, 1.5 nmol of dodecyl alcohol was performance liquid chromatography (HPLC) system added to the reaction mixtures, and then the mixtures were equipped with a CHIRALPAK IC 4.6 9 250 mm column extracted twice with Et2O or ethyl acetate. The reaction (Daicel, Osaka, Japan). The enantio-purity was confirmed products were concentrated by a stream of N2 and analyzed by chiral GC/MS with a GL Sciences InertCap CHIRA- by chiral and achiral GC/MS analyses as described above. MIX 30 m 9 0.25 mm column and 0.25 lm film. Helium was used as the carrier gas with a column head pressure of 100 kPa. The oven temperature was kept at 40 °C and Results increased with a gradient of 2.5 °C/min up to 180 °C. The chromatogram obtained with this analysis was shown in Chemical synthesis, chiral purification and determination of the absolute conformation of the hydroxyl group of IME A B Because the levels of the crude enzymatic products were too +0.16 -0.16 MTPA 7 MTPA 7 low to determine their enantiomeric properties by NMR, we O 1 O 1 H 6 H 2 6 2 -0.04 compared the synthesized IME and enzyme products. IME H +0.04 H H H H H was synthesized by Motherwell’s method [23]withslight +0.12 n.a. -0.12 4 -0.03 3 45 modification. The aluminum column chromatography gave 3 5 H H H H pure IME confirmed by normal GC, but it was shown by 9 10 chiral GC that this fraction contained two enantiomers 9108 8 -0.11 -0.05 n.a. n.a. (Fig. 1a upper). Each of the chiral compounds was enantio- purified by chiral HPLC. The absolute configurations of the Fig. 2 Determination of absolute configuration of the IME hydroxyl secondary hydroxyl group of IME shown as a retention time group that appeared at Rt = 40.8 min in Fig. 1 by the modification of Mosher’s method [24]. Dd values are displayed beside the protons. of 40.8 min in Fig. 1 were determined. The Dd values of Some protons in b were not assigned and displayed as n.a. positions 7 and 6 were plus values, and those of positions 3, 123 J Wood Sci (2014) 60:446–452 449

9, and 10 were minus values (Fig. 2a). Therefore, the C. lusitanica cells. The enantio-purity and the configura- absolute configuration of the hydroxyl group of this com- tion of authentic EMO had already been confirmed by pound was determined as having an S configuration NMR spectrum obtained with the modified-Mosher method (S-IME). Other enantiomer, Rt = 40.7 in Fig. 1, was also [14]. As shown in Fig. 4, the retention time on chiral GC of confirmed as having a R configuration (R-IME) (Fig. 2b). the authentic enantio-pure EMO and those of the crude enzyme-produced EMO were identical. And mass spectra Enantio-selective formation of IME by crude enzyme of both were also identical (data not shown). No other peak solution from C. lusitanica cultured cells with the same or similar mass spectrum as EMO was observed on chiral GC. It was, therefore, shown that the Chemically synthesized IME has two enantio configura- crude enzyme product was enantio-pure (1R,2S,6S)-(?)- tions on its hydroxyl group. To determine the enantiomeric 1,6-epoxy-4(8)-p-menthen-2-ol (RSS-EMO). purity of enzymatic products, we performed an assay of the terpinolene reaction with the crude microsomal fraction, and it was observed that the enzyme product gave a single peak at the same retention time as the S configuration A (Fig. 1a) of the authentic IME, which was purified enan- tiomerically and whose absolute configuration was determined as described above. Their mass spectra were also with S-IME substrate identical (Fig. 1c, d). Another configuration product,

R-IME, was not detected at this GC sensitivity; i.e., the Ion intensity enantiomeric excess value of this enzyme product was almost 100 %. It should thus be noted that the enantio- with R-IME substrate regulation by this enzyme was very strict.

10.5 11.0 11.5 12.0 12.5 13.0 13.5 Stereo substrate speciﬁcity of IME during EMO Retention time (min) formation

107 135 The subsequent metabolic step after IME formation was B epoxidation from IME to EMO [18]. The production of 67 only an S configuration by cytochrome P450 monooxy- 91 genase reaction with the C. lusitanica microsomal fraction 121 150 raised the next question about the substrate specificity of 168 another cytochrome P450, which oxidize IME. We, therefore, used enantio-pure synthesized IMEs as the next microsomal reaction. 60 80 100 120 140 160 EMO was not detected on GC/MS with the substrate of 107 R-IME at the level of our detection limit, but the C. lusi- 135 tanica microsomal reaction with S-IME gave the product, C EMO (Fig. 3a). The substrate specificity of this reaction 67 was thus very strict stereo-selective, accepting only S-IME. 91 121 In this experiment, no EMO produced by two succeeding 150 Ion intensity Ion intensity enzyme reactions was observed, probably because the 168 shorter reaction time we used in this experiment was not enough to detect EMO from the enzyme-produced IME. 60 80 100 120 140 160 m/z Enantio-purity of EMO produced by cytochrome P450 reaction Fig. 3 GC/MS analysis of EMO produced by the microsomal reaction with enantio-pure IME. The peak at 10.6 min is IME fed We also investigated the absolute configuration of EMO as a substrate. a Total ion chromatogram. Upper line the product with produced by this cytochrome P450-catalyzed oxidation. S-configuration IME. Lower line the product with R-configuration IME. The standard EMO appeared at 12.1 min. b The EI mass The crude enzyme product was compared on the chiral GC spectrum of standard EMO. c The EI mass spectrum of the peak at analysis with authentic EMO, which was obtained from the 12.1 min of the enzyme product

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cells, medium, or headspace air of cultures [14, 15]. HO O Another possibility is that the system might be exclusive to exogenous compounds including R-IME, but further observation is required to identify the role(s) of this stereo- RSS-EMO selectivity.

Authentic sample There are reports in which multiple products were Ion intensity generated in succeeding reactions by one cytochrome P450 [29–31] and in which regio-isomers were produced from Enzymatic product multiple substrates [32]). The possibility of succeeding 29 30 31 32 33 two-step reactions with one enzyme in our cell line was Retention time (min) low because only IME (no EMO) was observed in our in vitro enzyme reaction. Fig. 4 Chiral GC/MS analysis of EMO produced by the microsomal It has been reported that no products other than EMO cytochrome P450 prepared from C. lusitanica cells by selected ion monitoring on m/z = 168. Upper line selected ion chromatogram and no EMO diastereomer were found during EMO for- (m/z = 168) of standard EMO obtained from cultured cell. Enantio- mation by the crude enzyme [18]. In addition, we did not purity and configuration had been confirmed by Matsunaga et al. [14]. observe enantio-isomers other than RSS-EMO, and R-IME Lower line selected ion chromatogram (m/z = 168) of products by the substrate was not accepted in this EMO-forming reaction. microsomal cytochrome P450 from C. lusitanica cells. Any other peak than Rt = 31.35, including Rt = 32.1, did not have the same or The conformation of the hydroxyl group in EMO seems no similar mass spectrum as EMO, indicating production of a single difference from that of the original IME, and epoxy oxygen chiral product configuration by this assay was inserted from a specific direction; thus, the configuration of the hydroxyl group of IME is a very important property for substrate recognition.

Discussion Monoterpene There is a large family of a variety of cytochrome P450s synthase [1], and the variety is one of the reasons for the huge Geranyldiphosphate diversity of plant secondary metabolites. Among the secondary metabolites, terpenoids are the largest group of compounds; therefore, the research on the cytochrome HO P450s for plant terpenoids metabolism is keenly ongoing Terpinolene [25]. The first step of terpenoid synthesis is conducted by terpene synthases, which produce olefin terpenes and determine the basic skeletons. After that, skeletal inter- HO mediates are oxidized by cytochrome P450s [26, 27], and the biological activities of the final products strongly depend on how the molecule was modified. Moreover, O O O enantio differences present a critical issue in light of their HO HO HO biological activities for plant survival strategies and potential clinical uses in humans [4]. S-IME In our previous report [18], terpinolene was oxidized to IME and EMO by the cytochrome P450 in microsomal O fraction of C. lusitanica cells. Details of the stereo-selec- HO OH tivity of these terpinolene metabolisms were investigated in the present study. We found that IME produced by the O microsomal fraction with terpinolene had only an S configuration (Figs. 1, 2). The enantiomeric excess (e.e.) value -Thujaplicin was 100 % at our detection level; i.e., very strict stereo- RSS-EMO selectivity was observed in the formation of IME. S-IME may have a special role in C. lusitanica survival in nature, Fig. 5 Summary of stereo-selective oxidations from terpinolene by cytochrome P450s in C. lusitanica cultured cells. The pathway with considering the examples of the different biological solid arrows shows the metabolic pathway deduced by the experi- activities of each enantiomer [28]. However, this idea ments described in the main text. Others pathways in shaded area are conflicts with the finding that no IME was detected in the reactions not observed in the present study

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As described above and previously reported, this terpi- 12. Yamaguchi T, Fujita K, Sakai K (1999) Biological activity of nolene metabolism in C. lusitanica shows very strict sub- extracts from Cupressus lusitanica cell culture. J Wood Sci 45:170–173 strate-, regio- and stereo-selective reactions as summarized 13. Yamada J, Fujita K, Sakai K (2003) Effect of major inorganic in Fig. 5. nutrients on b-thujaplicin production in a suspension culture of One of the future goals of our project is to find a novel Cupressus lusitanica cells. J Wood Sci 49:172–175 pathway to form a tropolone ring such as b-thujaplicin. We 14. Matsunaga Y, Fujita K, Yamada J, Ashitani T, Sakai K (2003) Monoterpenes produced by Cupressus lusitanica cultured cells have already established a feeding experiment system with including a novel monoterpene (1S,2S,6S)- (?)-1,6-epoxy-4(8)- d6-terpinolene [17]. This system will reveal intermediates p-menthen-2-ol. 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